Atherosclerosis involves endothelial cells, smooth muscle cells (SMCs), macrophages, and T lymphocytes, but the individual contributions of these cell types to atherosclerotic plaque formation remains largely enigmatic. The goal of this project is to examine the contribution of the arterial wall to atherosclerosis, and to develop an approach for studying the genes important to this process. Three (3) such genes are those encoding tumor necrosis factor-a (TNF) and the 2 known receptors for TNF, an inflammatory cytokine secreted by both macrophages and SMCs. The importance of TNF in atherogenesis is highlighted by its role in promoting the secretion of C-reactive protein, serum levels of which are the best single predictors of incident myocardial infarction. To test the hypothesis that SMC TNF receptor-mediated signaling contributes significantly to atherogenesis, we will use 2 model systems. First, our in vivo model will use carotid interposition grafting in apolipoprotein E-deficient (Apoe-/-) mice, which develop carotid artery atherosclerosis. The grafts will be carotid arteries derived from congenic mice that are either (a) wild type (negative controls), (b) TNF receptor-1-deficient (Tnfr1-/-), (c) Tnfr2-/-, or (d) Tnfr1-/-.Tnfr2-/-. By comparing the atherosclerosis time course, extent, and plaque cellular composition in each of these grafts, we propose to assess the role of arterial wall TNFRs in atherogenesis. Second, our in vitro model of atherosclerosis will use macrophage/SMC co-cultures to assess the role of SMC and macrophage TNFRs in activated macrophage elicited gene expression and atherogenic activities of SMCs: proliferation, migration, and scavenger receptor activity. Both macrophages and primary aortic SMCs for co-culture will derive from each of the mouse lines described above, and macrophages will be activated by oxidized low-density lipoprotein. Thus, this project will (i) create a model system that can test whether specific genes contribute to arterial wall-mediated atherogenesis; (ii) elucidate the roles of arterial wall TNFRs in atherogenesis; and (iii) discern SMC genes expressed in response to factors secreted by activated macrophages. In so doing, this project should build a foundation for identifying multiple arterial wall gene products that may serve as therapeutic targets for atherosclerosis.